Nucleic acid encoding calcyon, a D-1 like dopamine receptor activity modifying protein

ABSTRACT

A number of cDNA clones whose products may interact with D1 receptors in vivo were identified. One of the clones, P24, was characterized further. P24 is localized in dendrites and spines of pyramidal cells in PFC. The extent of overlap between P24 expressing and D1 receptor expressing pyramidal cells appeared to be 100%. In contrast, only a limited number D1 receptor antibody labeled neurons in caudate expressed P24. P24 lowers the threshold of D1 receptor response to dopamine (DA) by an order of magnitude. Sequence similarity suggests P24 is a diverged member of the RAMP family. The P24 protein is therefore referred to as a D1 DA RAMP, calcyon. The isolated protein and nucleotide molecule encoding the protein, as well as primers for the nucleotide, are described. The protein and compounds modifying DA binding to the receptor or calcium release which is mediated by the Calcyon, are useful in research studies, drug screening, and therapeutically.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. patent application Ser. No.60/103,786, filed Oct. 9, 1998 and U.S. patent application Ser. No.60/130,609, filed Apr. 22, 1999. This application is a divisional ofU.S. patent application Ser. No. 09/416,509, filed Oct. 8, 1999, nowU.S. Pat. No. 6,469,141. U.S. patent application Ser. No. 60/103,786,filed Oct. 9, 1998, U.S. patent application Ser. No. 60/130,609, filedApr. 22, 1999, and U.S. patent application Ser. No. 09/416,509, filedOct. 8, 1999, are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

This is in the general area of protein receptors, and more specificallyrelates to isolation and cloning of a D1-like receptor activitymodifying protein (RAMP) which is a single transmembrane protein,designated P24, or calcyon, that interacts with a C-terminalintracellular segment of the hD1 DA receptor, and use thereof inscreening for therapeutics and diagnostics.

The human D1 dopamine (DA) receptor displays micromolar affinity for theneuromodulator DA. Currently, the management of many psychiatric andmovement disorders relies heavily on the inhibition or facilitation ofDA at its receptors. Via DA receptors, DA and DA mimetic ligands, suchas antipsychotic drugs, exert both short and long term changes in ionchannel activity, protein kinase/phosphatase activities, and geneexpression (Artalejo et al., Nature 348:239–42 (1990); Steiner andGerfen, J. Comp. Neurol. 353:200–12 (1995); Surmeirer et al., Neuron14:385–97(1995)). The design of most of these drugs has been based on a“D1” and “D2” DA receptor subtype paradigm. However, molecular cloninghas led to the identification of five mammalian DA receptor subtypes(D1–D5) (reviewed in Gingrich and Caron, Annu. Rev. Neurosci. 16:299–321(1993)). Individual subtypes have been further characterized as D1-like(D1 and D5) or D2-like (D2, D3 and D4) based on their selectivity foreither classical “D1” or “D2” dopaminergic ligands. Thus,conceptualizations of how DA modulates the mesolimbic, mesocortical andnigrastriatal pathways through “D1” and “D2” receptors are inadequategiven the added molecular complexity of the DA receptor system. Thefunctional implications of these “new” “D1” and “D2” DA receptorsubtypes for DA in regulating cognitive, motor and associative functionsis currently unknown. However, their discovery presents newopportunities for obtaining a more precise understanding of the role ofdopaminergic neurotransmission in these processes. In addition, acomplete understanding of the cellular and molecular processesregulating the specific subtype functions is crucial for dealing withdisorders like schizophrenia, Parkinson's disease, Tourette's syndrome,and drug addiction that appear to involve dysfunction in the DA system.

In brain, D1 receptors are most abundant in the caudate nucleus, wherethey are involved in the control of movement. D1 receptors are alsofound in prefrontal cortex (PFC) where they are required for workingmemory, a form of memory impaired in schizophrenia. In PFC, D1 receptorsare present in pyramidal cell dendritic spines typically located severalmicrometers away from DA terminals.

Immunohistochemical analyses of the D1-like dopamine (DA) receptorsubtypes, D1 and D5, shows that each receptor protein has a uniquecellular and subcellular distribution within the mesocortical,mesolimbic, and nigrastriatal pathways. These results support the notionthat each D1-like subtype serves a distinct function. However, themolecules that may mediate subtype-specific signal transductiondifferences in vivo have not yet been identified. In addition, detailsregarding the processes specifying the subcellular distribution of eachreceptor subtype are unclear. Without this molecular information, it isdifficult to understand the physiologic requirements for multipleD1-like subtypes. Examination of well-characterized systems indicatesthat most processes in cells are mediated by protein complexes createdby specific protein-protein interactions (Formosa et al., In: Methods inEnzymology: Academic Press, Inc. pp 24–45 (1991)).

As a group, the five mammalian DA receptor subtypes comprise a subfamilyof the G-protein coupled receptor (GPCR) superfamily with predictedseven transmembrane topology. Similarities in genomic organization,sequence, and G-protein coupling suggest that the two DA receptorfamilies evolved from prototypical D1 and D2 receptor genes viaduplication and divergence (O'Dowd et al., In Handbook of Receptors andChannels: CRC Press, Inc., pp 95–123 (1994)). Although each of theD1-like and D2-like subtypes are presumed to serve distinct functions,subtype-specific signal transduction differences have not beenidentified in vivo. Without this functional information, it is difficultto understand the physiologic requirements for multiple D1-like andD2-like receptor subtypes. Using the D1 and D5 D1-like subtypes as amodel, one can elucidate the physiologic basis for multiple DA receptorsubtypes by defining molecular determinants of their functions.Information obtained from this research should lead to a moresophisticated understanding of the principles guiding the functionalorganization of dopaminergic pathways. Several classes of proteins thatregulate GPCR's are known, yet none have been found to alter thesensitivity of D1 receptors in vivo.

There is ample pharmacological, electrophysiological and behavioralevidence to testify to the importance of D1-like receptors in cognitiveand motor processes under normal or pathological conditions, includingtardive dyskinesia (Ellison and See, Pyschopharmacology 98:564 (1989);Spooren et al., Euro. J. Pharmacol. 204:217 (1991)), Parkinson's Disease(Gilmore et al., Neuropharmacology 34:481–8 (1995)), working memory(Sawaguchi and Goldman-Rakic, Science 251:947–50 (1991)), and long termpotentiation (Huang and Kandel, Proc. Natl. Acad. Sci. USA 92:2446–50(1995)). However, whether the primary D1-like receptor involved is D1,or D5, or both, in each of these processes/behaviors is completelyunclear because both subtypes have similar affinities for “D1” receptoragonists and antagonists. mRNA and protein localization studies inrodent and primate have provided the most revealing insights into thedifferent functions of the D1 and D5 subtypes in vivo (Huntley et al.,Mol. Brain Res. 15:181–8 (1992); Levey et al., Proc. Natl. Acad. Sci.USA 90:8861–5 (1993); Smiley et al., Proc. Natl. Acad. Sci. USA91:5720–4 (1994); Laurier et al., Mol. Brain Res. 25:344–350 (1994);Bergson et al., J. Neurosci. 15:7821–36 (1995)). Although the D1 subtypeis the most abundant DA receptor subtype, many aspects of the D5subtype's localization in cerebral cortex and limbic nuclei suggest thatit may support DA's actions in the higher cognitive, associative andaffective processes uniquely associated with humans. In contrast, themost abundant expression of D1 receptors is detected in the basalganglia nuclei which are primarily associated with movement.

Previous studies carried out with subtype-specific antibodies indicatethat D1 and D5 receptor proteins are typically coexpressed in pyramidalneurons of monkey prefrontal cortex. However, initial electronmicroscopic studies suggest that D1 receptors are preferentiallylocalized in spines of pyramidal neurons, and D5 receptors are mainlyassociated with their apical dendrites (Bergson et al., J. Neurosci.15:7821–36 (1995)). As the synaptic input to spines is excitatory, andsynaptic input to shafts is generally inhibitory (Jones, Cerebral Cortex3:361–72 (1993); Harris and Kater, Annu. Rev. Neurosci. 17:341–71(1994); Smith et al., J. Comp. Neurol. 344:1–19 (1994)), it seemsreasonable to speculate that the two D1-like receptors may, in fact, becarrying out different functions. Their differential localization inpyramidal cell dendritic spines and shafts is consistent with the ideathat D1 and D5 receptors initiate biochemical events that modulateexcitatory or inhibitory synaptic transmission, respectively. Thispossibility has been supported by numerous electrophysiological studies(Cepeda et al., Proc. Natl. Acad. Sci. USA 90:9576–80 (1993); Cameronand Williams, Nature 366:344–7 (1993); Taber and Fibiger, J. Neurosci.15:3896–904 (1995)). Indeed, a recent electrophysiological studydemonstrated that DA's normal ability to potentiate responses to NMDA isblunted in D1 knockout mice (Levine et al., J. Neurosci. 16:5870–82(1996)).

D1 and D5 receptors, like a number of other GPCRs expressed in brain,stimulate adenyl cyclase in the presence of agonist presumably viacoupling to a Gs-like G-protein. However, knowledge of whether D1 or D5subtypes elicit specific physiological responses in vivo is lacking. Itis also unclear whether receptor-specific regulatory steps exist tomodify D1 versus D5 receptor activation. This molecular information iscritical for developing therapies that might inhibit or activate a D1 orD5 specific function.

It is an object of the present invention to provide reagents which canbe used to identify determinants which may permit the D1 and D5 subtypesto elicit unique cellular responses.

SUMMARY OF THE INVENTION

A number of cDNA clones whose products may interact with D1 receptors invivo were identified. One of the clones, K37, was characterized further.The protein (P24) encoded by K37 is localized in dendrites and spines ofpyramidal cells in PFC. The extent of overlap between P24 expressing andD1 receptor expressing pyramidal cells appeared to be 100%. In contrast,only a limited number of D1 receptor antibody labeled neurons in caudateexpressed P24. P24 lowers the threshold of D1 receptor response todopamine (DA) by an order of magnitude. Sequence similarity suggests P24is a diverged member of the RAMP family. The P24 protein is thereforereferred to as a D1 DA RAMP, and was given the name Calcyon. Theisolated protein and nucleotide molecule encoding the protein, as wellas primers for the nucleotide, are described. The protein and compoundsmodifying DA binding to the receptor or calcium release which ismediated by Calcyon are useful in research studies, drug screening, andtherapeutically.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is the nucleotide sequence, SEQ ID NO: 1and amino acid sequence(SEQ ID NO: 2) of a D1-like dopamine receptor activity modifyingprotein. Optimal Kozak sequence for initiation by eukaryotic ribosomesis shown in bold letters. Putative transmembrane domains are shadedgray. A putative N-linked glycosylation site is marked with an asterisk.Putative protein kinase C phosphorylation sites are overlined.

FIG. 2 is an amino acid sequence alignment of Calcyon (P24) and neuronaldendritic proteins P19 (SEQ ID NO: 6) and P21 (SEQ ID NO: 7).

FIGS. 3 a, 3 b, and 3 c are graphs showing that calcyon inhibits P2Y andM1 receptor stimulated Ca⁺⁺ _(i) release. Ligand induced Ca⁺⁺ release isshown for FURA-2-loaded D1 HEK293 cells expressing EGFP (FIG. 3 a) orEGFP-Calcyon (FIGS. 3 b and 3 c). Substances were applied for timesindicated by horizontal bar. Ca⁺⁺ signals are reported as the mean of6–8 cells. Similar results were obtained from at least two additionaltransfection experiments.

FIG. 4 is a graph showing that calcyon's cytoplasmic domain binds PIP2.S-Calcyon bound to 25 μl of S-agarose resin, or 25 μl of S-agarose resinonly was incubated for one hour at room temperature with phospholipidvesicles containing phosphotidylcholine (PC), phosphotidylethanolamine(PE), or PC, PE, and PIP₂. Both types of vesicles were radioactivelylabeled with equivalent amounts of ³H-phosphatidylcholine. Followingincubation, resins were washed three times with ten volumes of buffer(20 mM hepes, pH 7.4), and amounts of bound phospholipid determined byliquid scintillation counting. Results are reported as percent of inputradiolabel precipitated. Bars represent average value obtained in threeindependent assays with standard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

I. Proteins and Processes Involved in D1 and D5 Receptor Function andSubcellular Localization

Neurotransmitter receptors are typically clustered at postsynapticmembrane specializations opposed to presynaptic nerve terminals. Recentstudies of the nicotinic acetylcholine (nAch), NMDA, and glycine (Gly)receptors indicate that protein:protein interactions may be important intethering these receptors to the postsynaptic membrane. For example, a43 kDa, zinc finger containing a protein called rapsyn appears to beabsolutely essential for Ach receptor clustering at the neuromuscularjunction (Froehner et al., J. Cell Biol. 114:1–7 (1991)). Rapsyn inducesAch receptor clustering when expressed in heterologous cells (Phillipset al., Science 251:568–70 (1991)); and, in rapsyn knockout mice, Achreceptors do not cluster anywhere within muscles (Gautam et al., Nature377:232–6 (1995)). Likewise, clustering of Gly receptors at synapticspecializations requires accumulation of a protein expressed throughoutthe CNS called gephyrin (Kirsch et al., Nature 366:745–8 (1993)).Gephyrin copurifies with Gly receptors and also appears to associatewith cytoskeletal elements (Kirsch and Betz, J. Neurosci. 15:4148–56(1995). Recently, the NR2 subunit of NMDA receptors was found tointeract with a postsynaptic density guanylate kinase called PSD-95. Theinteraction between PSD-95 and NR2 may serve to anchor NMDA receptors atthe postsynaptic membrane, or assemble a multienzyme complex involved inNMDA-receptor mediated synaptic plasticity (Kornau et al., Science269:1737–40 (1995)). It is not yet known if proteins analogous togephyrin, rapsyn or PSD-95 are required for the synaptic localization ofD1 or D5 receptors. On the other hand, how the D1-like receptors aredirected to synapses within different membrane compartments (e.g., anapical dendritic shaft vs. spine) may involve cytosolic proteins. Suchproteins may chaperone newly synthesized proteins to different areas ofthe plasma membrane, rather than serve as their “receptors” already inplace at a given postsynaptic specialization (Casey, P. J., Science268:221–5 (1995)).

Studies of the β₂-adrenergic receptor and rhodopsin indicate that GPCRsare rapidly inactivated following agonist binding, and removed from theplasma membrane to endosomes, where they are resensitized. In large partthis process is mediated by two families of proteins that interact withthe C-terminal tail of GPCRs. One family includes the GPCR kinases thathave been found to phosphorylate rhodopsin and the β₂-adrenergicreceptor at C-terminal serine and threonine residues.

The other class of proteins involved in receptor desensitization iscalled the arrestins. Arrestins bind GPCR kinase-phosphorylatedreceptors and play a key role in their removal from the plasma membrane.It has been proposed that arrestins may recruit other proteins involvedin relocating receptors to endosomes, or may activate this movementthemselves (Ferguson et al., Science 271:363–6 (1996)). Two arrestinproteins that interact with Drosophila rhodopsin exhibit different ratesof rhodopsin inactivation suggesting multiple arrestin molecules may berequired to regulated different levels of GPCR activation (Dolph et al.,Science 260:1910–6 (1993)). Identification and characterization of GPCRkinases and arrestins that interact with the D1 and D5 DA receptors mayreveal differences in D1-like receptor desensitization in vivo.

Electrophysiological studies of dissociated neostriatal medium spinyneurons suggests that D1-like receptor agonists stimulate a cascade ofkinase and phosphatase activity that ultimately result in reduced N- andP-type Ca⁺⁺ currents (Surmeier et al., Neuron 14:385–97 (1995)). Thesubcellular localization of a variety of kinases and phosphatases fromorganisms ranging from yeast to mammals is determined by “scaffold” and“anchoring” proteins that appear to physically bring together componentsof coordinated, complex, signaling events. “Scaffold” proteins appear tosimultaneously associate with several components of a signaling pathway(Faux and Scott, Cell 85:9–12 (1996)). For example, pheromones whichinitiate mating in yeast bind to a GPCR which results in activation of acascade of five different kinases. The yeast protein Ste5p representsthe model scaffold protein as it associates with each of these kinases.Anchoring proteins are tethered to submembranous or cytoskeletalstructures, and localize a group of signal transduction enzymes to theirsite of action (Mochly-Rosen, Science 260:1910–6 (1995). In neurons,type II cAMP dependent protein kinase (PKA) is localized to postsynapticdensities by its interaction with A-Kinase Anchoring Protein, AKAP79.Besides PKA, AKAP79 binds Ca++ calmodulin-dependent protein phosphatase2B, calcineurin, and α and β isoforms of protein kinase C (Coghlan etal., Science 267:108–11 (1995); Klauck et al., Science 271:1589–92(1996)). Both scaffold and anchoring proteins compartmentalizefunctionally related enzymes, and are therefore poised to tightlyregulate signaling pathways through protein-protein interactions. It ispossible the kinases and phosphatases involved in D1-like receptormodulation of Ca⁺⁺ channels, and D1-like receptor may be associated witha common scaffold or anchoring protein.

II. Identifying Molecules That Interact with D1-Like Receptors

Like many cellular processes including gene transcription and synapticvesicle targeting, there is an emerging picture that the cascade ofintracellular events set off by neurotransmitter receptor activationrequires large assemblies of proteins constructed of specificprotein-protein interactions (Formosa et al., In: Methods in Enzymology:Academic Press, Inc. pp 24–45 (1991); Rothman, J. E., Nature 63:55–63(1994)). By analogy with other neurotransmitter receptors, it ishypothesized that the proteins with which the D1-like receptors interactin vivo specify some aspect of D1 or D5 subtype-specific signaling orsubcellular localization. Immunohistochemical analyses of the D1 and D5receptors in the primate brain indicated that the two mammalian D1-likeDA receptors may be serving subtype-specific functions due to theirunique cellular and subcellular distributions (Bergson et al., J.Neurosci. 15:7821–36 (1995)). Molecular and cellular processes thatdifferentially regulate D1 and D5 receptor subtype function andsubcellular localization in vivo have since been identified. Thisresearch has progressed steadily as the result of developing thefollowing tools: (1) yeast two-hybrid system for detection and analysisof D1 and D5 receptor protein:protein interactions; (2) subtype-specificmonoclonal antibodies for immunochemical detection of D5 receptor:protein interactions; and (3) inducible DA receptor expression in stablytransfected mammalian cells for subcellular localization analysis,ligand binding, and adenylyl cyclase assays studies. These studiesprovide the means to determine the requirements for multiple D1-like andD2-like subtypes.

Calcyon

Calcyon, having the amino acid (SEQ ID No. 2) shown in FIG. 1, is anexample of a protein that may interact with the D1 and D5 receptors,isolated using this system.

Calcyon interacts with D1 DA receptors and enables the typicallyG_(s)-coupled D1 receptor to stimulate robust Ca⁺⁺ _(i) release. Theresults indicate that in the presence of Calcyon, D1 receptors cansimultaneously activate both Ca⁺⁺ and cAMP-dependent signaling pathways.The mechanism underlying Calcyon enabled D1 receptor stimulated Ca⁺⁺_(i) release appears to be independent of cAMP, as Calcyon expressiondoes not alter D1 receptor-stimulated cAMP accumulation. The small risein Ca⁺⁺ _(i) observed following D1 receptor stimulation in the absenceof Calcyon may result from cAMP-dependent protein kinase (PKA)enhancement of IP₃ receptor-induced Ca⁺⁺ mobilization (Wojcikiewicz, R.J. H., et al., J. Biol. Chem 273:5670–7 (1998)). Such an indirectmechanism is likely to be temporally out of synchrony with the immediateburst of Ca⁺⁺ mobilized by D1 receptors in the presence of Calcyon.However, the mechanism likely involves binding of IP₃ to IP₃ receptorslocated on vesicular Ca⁺⁺ stores. The most direct means of generatingIP₃ involves phospholipase C (PLC)-catalyzed hydrolysis ofphosphatidylinositol 4,5-bisphosphate (PIP₂) to IP₃ and diacylglycerol(DAG). Stimulation of PLC is a response known to be mediated by theG_(q/11) class of G proteins (Hamm, H. E., J. Biol. Chem 273:669–72(1998)). Thus, it is believed that Calcyon interaction facilitates D1receptor coupling to members of the G_(q/11) family of G proteins,without disabling receptor coupling to G_(S). Site-specific mutagenesisof the G_(q)-coupled rat M3 muscarinic receptor revealed that fourresidues (Arg252-Ile253-Tyr254-Lys255) (SEQ ID NO: 8) near theN-terminus of the third intracellular loop (i3) are critical forefficient G_(q/11) activation and stimulation of PIP₂ hydrolysis (Bluml,K., et al., J. Biol. Chem 269, 402–5 (1994)). This sequence is alsopresent in the analogous region of the G_(q)-coupled M1 and M5 receptors(Blin, N., et al., J. Biol. Chem 270:17741–8 (1995)). The D1 receptorcontains an almost identical sequence (Arg216-Ile217-Tyr218-Arg219) (SEQID NO: 9) located at the N-terminus of i3.

Calcyon-enabled, D1 receptor-stimulated Ca⁺⁺ release occurs after prioractivation of a G_(q) coupled GPCR. G_(q)-coupled GPCR stimulation leadsto PKC activation via DAG and Ca⁺⁺ generation (Oancea, E., et al., Cell95:307–18 (1998)). Although Calcyon is phosphorylated in unstimulatedcells, the level of Calcyon phosphorylation increases followingtreatment with the PKC activator, PMA. Therefore, G_(q) coupled GPCRactivation may be necessary to stimulate PKC-dependent phosphorylationof Calcyon. PKC inhibition prevents Calcyon-enabled, D1receptor-stimulated Ca⁺⁺ mobilization, consistent with this idea. BLASTsearch of the SwissProt database using the predicted intracellulardomain of Calcyon revealed significant similarity between thealanine-rich region of Calcyon and the myristolated alanine-rich Ckinase (PKC) substrate (MARCKS) protein. MARCKS has been shown toinhibit PLC-catalyzed hydrolysis of PIP₂ by sequestering PIP₂ (Glaser,M., et al., J. Biol. Chem 271:26187–93 (1996)). Because Calcyonexpression decreases P2Y and M1 receptor-stimulated Ca⁺⁺ _(i) release,the results are consistent with the possibility that Calcyon may alsosequester PIP₂. PKC dependent phosphorylation of MARCKS releasessequestered PIP₂. Reasoning by analogy, if G_(q)-coupled receptorstimulation leads to additional phosphorylation of Calcyon, theliberated pools of PIP₂ may contribute to the large increase in Ca⁺⁺mobilized by D1 receptor stimulation.

Calcyon is a single transmembrane protein that exhibits extensivesequence identity with the neuronal dendritic proteins, P19 and P21.Recently, another family of single transmembrane proteins calledreceptor activity modifying proteins (RAMP) has been reported toregulate calcitonin-receptor-like receptor (CRLR) function (McLatchie,L. M., et al., Nature 393:333–9 (1998)). Peptide hormone GPCRs, likeCRLR, interact with ligands through their N-terminal extracellularsegments (Ji, T. H., et al., J. Biolog. Chem 273, 17299–302 (1998)).Presumably, association with the extensive extracellular domains of theRAMP family alters CRLR affinity for the calcitonin-related peptides,CGRP and adrenomedullin. On the other hand, GPCR cytoplasmic domains arecrucial for specifying intracellular signaling pathways (Hamm, H. E., J.Biol. Chem 273, 669–72 (1998)). The data suggests that, via cytoplasmicdomain interactions, Calcyon expands the repertoire of signalingpossibilities for the D1 DA receptor. As such, RAMPs and Calcyon bothappear to modify GPCR function, but through different mechanisms. TheRAMP and P19/21/24 family of proteins display extensive sequencesimilarity within their predicted transmembrane segments suggesting thatCalcyon is a diverged member of the RAMP family. Since D1 DA RAMP(Calcyon) is more similar to P19 and P21 proteins than to RAMP1, 2 and3, it is proposed that the family of G-protein coupled RAMPs nowincludes at least two subfamilies.

The mechanism by which Calcyon alters D1 receptor and G_(q)-coupledreceptor function in HEK293 cells provides a molecular framework fortesting how receptors for DA, as well as for other neurotransmitters,modulate the actions of other neurotransmitters and hormones. As Calcyonis a member of a larger protein family, it seems possible that P19 andP21 may also act as ‘molecular bridges’ between GPCR signaling pathways.It is also possible that other neurotransmitters that regulate PKC may‘prime’ the D1 receptor-stimulated Ca⁺⁺ release enabled by Calcyon. Akey candidate is glutamate, as spines of pyramidal cells are the site ofexcitatory amino acid input. Stimulation of NMDA receptors leads toinflux in Ca⁺⁺ and can result in activation of Ca⁺⁺-dependent isoformsof PKC (Oancea, E., et al., Cell 95:307–18 (1998)) which is an important‘priming’ step in Calcyon activation. Numerous electrophysiological, aswell as molecular models of synaptic plasticity indicate that D1receptors influence NMDA receptor-mediated glutamate transmission(Cepeda, C., et al., Synapse 11:330–41 (1992); Cameron, D. J., et al.,Nature 366:344–7 (1993); Huang, Y. Y., et al., Proc. Natl. Acad. Sci.USA 92:2446–50 (1995); Williams, G. V., et al., Nature 376:572–5 (1995);Konradi, C., et al., J. Neurosci. 16:4231–9 (1996)). As Calcyonlocalizes to spines of pyramidal neurons in primate prefrontal cortex,it is poised to powerfully modulate excitatory transmission. D1(Bergson, C., et al., J. Neurosci. 15:7821–36 (1995)) and M1 receptors(Mrzljak, L., et al. Proc. Natl. Acad. Sci. USA 90:5194–8 (1993)) alsolocalize to spines of pyramidal neurons, raising the possibility thatthe mechanism of muscarinic/dopaminergic ‘receptor interaction’ definedhere may be relevant in vivo (Wang, J. Q., et al., J. Pharmacol. Exp.Ther. 281:972–82 (1997)). In particular, the role of Calcyon inpotentiating Ca⁺⁺ signaling pathways may provide insight into the D1receptor-dependent cognitive functions of prefrontal cortex that arecompromised in schizophrenia (Williams, G. V., et al., Nature 376:572–5(1995)).

Molecules that Alter Binding to Calcyon

The cDNA encoding Calcyon can be expressed in a variety of mammaliancell lines, including the fibroblast cell line described above, or inother commercially available-cell lines such as Cos cells, and used toscreen for compounds which bind specifically to the Calcyon. This isdetermined by comparing binding affinities for the various D₁, D₂, andD₃ receptors with that of Calcyon, then testing in vivo those compoundswhich specifically bind the receptor. It can also be expressed inbacterial cells, notably E. coli, as well as other eukaryotic expressionsystems such as Baculovirus infection of insect cells.

Compounds which bind either the human or the rat Calcyon can be screenedusing physiological models. The typical models for physiological testingof these compounds are rats, mice and dogs. Measurements can be made inintact animals, in tissue explants or in isolated cells.

The gene and/or cDNA can also be used to generate probes for screeningin a manner similar to those methods described above for receptors otherthan the known D₁, D₂, D₃, and D₄ dopamine receptors. Probes are createdfrom sequences generally fourteen to seventeen nucleotides in length,and can be labelled using available technology and reagents, includingradiolabels, dyes, tomography positron emission labels, magneticresonance imaging labels, enzymes, and fluorescent labels. Probes can beused directly or indirectly via standard methodologies includingpolymerase chain reaction (PCR) and methodologies to generate largerfragments of the Calcyon. Starting with either RNA (via RT PCR) or DNA,the Calcyon cDNA, and parts therein, can also be used to generate RNAtranscripts if cloned into appropriate expression vectors (cRNAs).

As used herein, a primer or probe said to hybridize specifically to aparticular nucleic acid fragment, sequence, or segment, or to a class ofnucleic acid fragments, sequences, or segments, refers to a primer orprobe that hybridizes to the particular nucleic acids and does nothybridize significantly to other nucleic acids present in the samesample under the hybridization conditions used. As used herein,significant hybridization refers to hybridization that is detectablewith the detection technique being used to detect specifichybridization. It is understood that some probes and primers willhybridize specifically to particular nucleic acids under somehybridization conditions but will not hybridize specifically to thosenucleic acids under different conditions. That is, the probe or primermay hybridize both to the particular nucleic acids and to other nucleicacids. Thus, a probe or primer that hybridizes specifically toparticular nucleic acids under at least one set of conditions is, asused herein, a probe or primer said to hybridize specifically to theparticular nucleic acids. A variety of hybridization conditions can beused to hybridize probes or primers to nucleic acids.

Calcyon DNA fragments, oligonucleotide probes or cRNAs, could all beused in commercial kits or sold separately to measure Calcyon transcriptlevels using standard techniques including PCR, in situ hybridization,and RNAse or SI protection assays.

Amino acid sequence can be deduced from fragments of Calcyon, or theentire Calcyon coding sequence, generated by a variety of standardtechniques for synthesis of synthetic peptides, Calcyon fusion proteinsand/or purification of Calcyon proteins (or parts thereof) from in vitrotranslated proteins derived from synthetic Calcyon RNA or proteinpurification per se. Calcyon proteins, peptides, fusion proteins orfragments thereof could subsequently be used for antibody productionusing available technology including injection into a wide variety ofspecies including mice, rats, rabbits, guinea pigs, goats, etc. for theproduction of polyclonal antisera as well as injection into mice andsubsequent utilization of fusion techniques for the production ofmonoclonal antibodies.

Oligonucleotides or larger sequences derived from Calcyon mRNA orcomplementary sequences or antibodies directed against Calcyon could belabelled or derivatized to be used as imaging agents for positronemission tomography (PET) or magnetic resonance imaging (MRI) of thelocation of Calcyon in vivo and in vitro.

Diagnostic and Therapeutic Applications

As demonstrated in the examples, Calcyon plays a role in DA binding andcalcium release. Compounds which alter Calcyon activity or calcyonmediated activity, can therefore be used in research or in therapeuticapplications similar to those in which other RAMP proteins are utilized.These compounds can be identified as described above and in more detailbelow.

EXAMPLES

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1 Characterization of cDNA Clones Isolated in a Two-HybridScreen for Proteins that Interact with the hD1 Dopamine Receptor

Yeast two-hybrid systems are useful for detection and analysis of D1 andD5 receptor protein:protein interactions. The two-hybrid system,initially described in yeast Saccharomyces cerevisiae, is a powerfulmolecular genetic technique for identifying new regulatory molecules,specific to the protein of interest (Fields and Song, Nature 340:245–6(1989)). Typically, these regulatory proteins could not have beenidentified using previous biochemical techniques likeimmunoprecipitation or cofractionation. This method has successfully ledto the identification of proteins associated with NMDA receptor NR2subunits (Komau et al., Science 269:1737–40 (1995), Huntington's protein(Li et al., Nature 378:398–402 (1995)), Fragile X Mental RetardationSyndrome gene product (Zhang et al., EMBO J. 14:5358–66 (1995)), inaddition to the D2 long DA receptor (Pepperl et al., Soc. Neurosci. Ann.Mtg., San Diego Abstract 253:11 (1995)).

Two-hybrid screens are currently the most sensitive method foridentifying novel protein:protein interactions. Exploiting this methodrepresents a unique opportunity to identify proteins that interact withthe D1 and D5 receptors. If proteins are involved in their transport, orlocalization in spines and dendrites, then it seems reasonable to expectthat proteins which specify the differential subcellular localization ofD1 and D5 receptors may be identified. In addition, two-hybrid screensmay also identify novel heterotrimeric G-protein isoforms that mayspecify their differential coupling to the cAMP signal transductionpathway, or identify proteins that regulate that interaction, e.g. anovel receptor kinase or phosphatase. There are also a number ofbiochemical approaches for identifying proteins that interact with theD1-like receptors, including coimmunoprecipitation and affinitychromatography. A two-hybrid system for isolating and understanding themolecular and cellular determinants that contribute to the differentfunction and subcellular localization of the D1-like subtypes in vivoinvolves the following steps:

-   -   1. isolating positive cDNA clones isolated in a yeast two-hybrid        screen of a human brain GAL4 activation domain library with the        C-terminus of the D1 receptor and/or the C-terminus of the D5        receptor,    -   2. characterizing the isolated clones by determining the DNA        sequence of positive clones,    -   3. obtaining full-length cDNA clones,    -   4. determining the tissue and brain regional expression pattern        of positive clones,    -   5. co-localizing D1 and D5 receptors and positive clones in        brain sections and transfected cells, and    -   6. characterizing how interacting protein influences        coupling/binding/localization of D1 and D5 receptors.

Methods other than a two-hybrid system can also be used to identifymolecules that interact with the D1-like receptors. For example,immunoprecipitation or cofractionation have been used to isolatenumerous biological macromolecules. Affinity chromatography can also beused to isolate biological macromolecules. Standard genetic screens canalso be used in certain organisms, such as yeast, to isolate moleculesinvolved in metabolic pathways.

A general example of how an immunoprecipitation method could be used isas follows. Monoclonal antibodies to D5 receptors can be used to isolateinteracting proteins by coimmunoprecipitation. Molecules thatcoimmunoprecipitate can be purified and then microsequenced byperforming tryptic digests. This information can be used to developoligonucleotides which can be used to screen cDNA libraries to isolatepositive clones. These clones can then be characterized.

A general example of an affinity chromatography method is as follows. D5receptor fusion proteins immobilized to an agarose matrix can be used toisolate interacting proteins. The microsequence of the isolated proteinscan then be determined using tryptic analysis, and the protein sequenceinformation can be used to obtain oligonucleotide probes. These probescan be used to obtain cDNA clones. The cDNA clones can be isolated andsequenced. The interactions can also be functionally characterized.

I. D1 Receptor:GAL4 DNA Binding Domain Fusion Protein

Sequence encoding C-terminal 81 amino acids of the hD1 receptor(residues 365–446) was subcloned into the yeast GAL4 DNA binding domainplasmid pGBT9, creating pGBTD1 (Fields and Song, 1995). A 252 base pairEcoRI-Sal I fragment encoding the C-terminal 81 amino acids of the humanD1 receptor was inserted into the multiple cloning site of pGBT9 at the3′ end of the open reading frame for the GAL4 DNA binding domain.Transcription of the fusion gene is activated by the constitutive ADH1promoter, P_(ADH1); and terminated at the ADH1 transcription terminationsignal, T_(ADH1). The plasmid contains selectable marker trp1 for growthon media lacking tryptophan (-trp). Yeast strain Y190 cells transformedwith pGBTD1 were reacted with rabbit anti-D1 receptor antibodies andCy3-conjugated goat anti-rabbit secondary antibody, then stained withDAPI. Cy3 and DAPI fluorescence showed coincident labeling of yeastnuclei by both D1 antibodies and DAPI. Cy3 fluorescence was notdetectable in Y190 cells transformed with pGBT9 vector DNA, followingsimilar incubation with anti-D1 and Cy3-conjugated antibodies.

The D1/GAL4 BD fusion was further tested for its ability to activatetranscription of a lacZ reporter gene located on the Y190 chromosomeusing the X-Gal colony filter assay. pGBTD1 did not activateβ-galactosidase transcription alone, or if co-expressed with GAL4Activation Domain (AD) protein encoded on the vector pGAD424. Thenuclear localization, and lack of independent lacZ expression of theD1/GAL4 BD fusion protein confirmed that it would be possible to detectinteracting GAL4 AD fusion proteins.

Cotransformants of pGBTD1 and pGAD424 were streaked on SC-Trp, -Leumedia. pGAD424 carries the leu2 gene for selection. As a positivecontrol, Y190 transformed with yeast interacting proteins snf1 and snf4were also streaked on the plate (snf1+snf4). A nitrocellulose filter wasplaced on the plate to transfer the colonies to the filter. Cells werepermeabilized by freezing in liquid nitrogen, and then soaked in buffercontaining X-Gal. Colonies containing the D1/GAL4 BD fusion proteinremained cream colored, showing that the D1/GAL4 BD protein does notindependently stimulate β-galactosidase gene transcription. In contrast,the positive control colonies, snf1+snf4, turned deep blue. The resultsshow the lack of independent lacZ expression of the D1/GAL4 BD fusionprotein.

This segment of the D1 receptor exhibits <20% amino acid sequencesimilarity with the analogous region of the D5 receptor. The yeaststrain Y190 was transformed with pGBTD1 using the lithiumacetate/glycerol procedure (Chen et al., Current Genetics 21:83–4(1992)). pGBT9 contains the trp1 marker which allows selection forplasmid uptake. Transformed cells were selected for on SC-Trp medium.The D1:GAL4 fusion protein was localized in nuclei of Trp+transformedY190. Therefore, the D1/GAL4 BD fusion protein isappropriately localized for contact with candidate interacting proteinsfused to GAL4 Activation Domain (AD) in a two-hybrid screen.

II. Screen of Human Brain cDNA:GAL4 Activation Domain Library withpGBTD1.

A human brain cDNA library in the GAL4 activation domain vector, pACT2,was purchased from Clontech (Palo Alto, Calif.). This library consistsof 5.0×10⁶ independent clones with inserts ranging in size from 0.5–4.5kb. 500 μg pACT2 library plasmid DNA purified from 8×10⁶ recombinantcDNA clones was transformed into the pGBTD1 transformed Y190 describedabove (Allen et. TIBS, December, 1995, pp. 511–516). Transformantscontaining pGBT9 and recombinant pACT2 were plated on 50 150 mm platescontaining SC-Trp, -Leu media, grown for three days at 30°, and thenassayed for β-galactosidase activity. Blue colonies were taken eitherdirectly from the filter or from the original plate, streaked for singlecolonies and re-tested for β-galactosidase activity in the X-Gal filterassay. 25 Leu+,Trp+transformants exhibited detectable levels of lacZgene expression after three rounds of single colony cloning.Nitrocellulose lifts, β-galactosidase activity assays, and growth onleu-trp-his-plates in the presence of 30 mM and 100 mM3-amino-1,2,4-triazole were done as described by Durfee et al., GenesDev. 7:555–69 (1993). The nucleotide sequence of both strands of pACTP24insert DNA was determined using an ABI Automated DNA Sequencer. Proteinsequences were aligned using CLUSTALW program on the EBI, UK server, andBOXSHADE 3.21 program on the ISREC, Switzerland server.

III. Genetic Characterization of Interacting Activation Domain cDNAClones

To test whether lacZ expression depended on coexpression of the GAL4binding domain, the pGBTD1 plasmid was eliminated from these clones.Loss of pGBTD1 was accomplished by streaking single clones on rich YEPDmedium, and replica plating on SC-Trp, SC-Leu, and SC-Trp-Leu media.Colonies that grew on SC-Leu, but not on SC-Trp or SC-Trp, -Leu had lostthe TRP marker (pGBT9), but retained the library pACT2 plasmid. TheseLeu+ colonies were picked, streaked on SC-Leu medium, and assayed forβ-galactosidase activity. Of the 25 Leu+ clones picked, only oneexhibited GAL4 binding domain independent lacZ gene expression by theX-GAL filter assay. This clone was eliminated from further analysis.

Transcription of the his3 gene in yeast strain Y190 is regulated by theGAL4 upstream activator sequence. Therefore, D1:AD clone interactionscan also be “selected” for by plating on SC-Trp, -Leu, -His mediacontaining 3-aminotriazole (3-AT), a competitive inhibitor of histidine.100 mM is considered a stringent concentration of 3-AT, and 30 mM lessstringent. Comparison of growth on SC-Trp, -Leu, -His+100 mM 3-AT versusSC-Trp, -Leu, -His+30 mM 3-AT provides a preliminary means of evaluatingthe strength of interaction between activation domain clones and D1sequences. Eight of the 24 Leu+, Trp+ transformants were streaked on +30and +100 mM 3-AT media. Six of the eight grew equally well on SC-Trp,-Leu, -His+30 mM and +100 mM 3-AT plates indicating very stronginteraction between these clones and the D1 C-terminus. While the othertwo thrived on the +100 mM 3-AT plates, they grew better on +30 mM 3-ATplates indicating that the interaction may not be as strong.

The specificity of interaction between the recombinant AD clones and theD1 C-terminus was also tested by asking whether the AD clones interactwith unrelated GAL4BD fusions. AD clones were tested with p53, caseindependent kinase 2 (cdk2), and snf 1 GAL4 binding domain fusions.Leu+Y190 colonies containing recombinant AD clones corresponding toeight of the clones that grew on 3-AT were mated with Trp+Y 187 strainstransformed with either p53:, cdk2:, or snf1:GAL4 BD plasmids. None grewon SC-Trp, -Leu, -His +100 mM or 30 mM 3-AT media. Lack of growthindicates that these eight library clones do not interact with eitherp53, cdk2, or snf1, adding further confidence in the specificity ofinteractions with D1 C-terminal residues. The remaining sixteen ADclones were being tested for growth on -His +30 and +100 mM 3-AT media,and interaction with unrelated GAL4 BD fusions.

IV. Molecular Characterization of Interacting Activation Domain cDNAClones

Recombinant pACT2 activation domain plasmid DNA was purified from theabove eight Leu+Y190 and transformed into E. coli strain DH5 byelectroporation. The size of the cDNA insert was determined by PCR usingprimers complementary to 5′ and 3′ vector sequences flanking themultiple cloning site. Insert sizes ranged from 500 bp to 1.8 kb.

Partial nucleotide sequence of the eight candidate positive clones wasobtained using the ABI system at the Macromolecular Core Facility atPenn State College of Medicine. The sequence of the GAL4 activationdomain cDNA inserts was determined using an oligonucleotide primercomplementary to vector sequence located 5′ of the multiple cloningsite. A BLAST search of the GenBank, as well as the EST and STSdatabases was conducted with the DNA sequence of each insert. BLASTanalysis of some clones revealed sequence similarity with knownproteins, or with cDNA clones isolated in the human brain expressedsequence tag (EST) project. The sequences of other activation domainclones are novel. Although the homologies detected are based on DNAsequences obtained from sequencing one strand, several cDNAs seem toencode proteins with potentially relevant functions. For example, cloneK37 exhibits extensive sequence identity with a family of relatedproteins variously called 21K, human brain neuronal protein-1, and 19KGolgi protein (Sutcliffe et al., Cell 33:671–82 (1983); Saberan-Djoneidiet al., J. Biolog. Chem. 270:1888–93 (1995). The 19K Golgi protein isexpressed in the Golgi apparatus of neural cells (Saberan-Djoneidi etal., J. Biolog. Chem. 270:1888–93 (1995)).

Immunohistochemical studies with peptide antibodies to brain neuronalprotein-1 revealed labeling of many large projection neurons includingpyramidal neurons. Antibody reactivity was concentrated in cytoplasmwith a polarity that suggested that BNP-1 may be involved in synthesisof proteins destined for dendrites (Sutcliffe et al., Cell 33:671–82(1983)). The sequence of 21K has not been published, but is entered inthe GenBank database (accession #M98530). The 21K gene maps next to theHuntington's Disease marker D4S10, and shows homology with proteinphosphatase inhibitors.

Clone #24–29 exhibits extensive sequence identity to an EST clone thatis similar to a zinc finger protein called rhombotin-1, and torhombotin-1 itself. Rhombotin-1 is expressed mainly in the centralnervous system and thymus (Boehm et al.,Oncogene 6:695–703 (1991)). Inaddition, clone #24–29 exhibits sequence similarity with the cysteinerich zinc finger region, of two human lim domain proteins hLH-1 andhLH-2. The zinc finger region in these proteins, called a LIM domain, isthought to be involved in protein:protein interaction. A variety of LIMdomain proteins have been isolated. Functions associated with LIM-domainproteins include development, cytoskeletal structure, signaling andtrafficking, and growth control (Gill, G. N., Structure 3:1285–9(1995)). It may also be noteworthy that rapsyn is also a zinc fingerprotein (Froehner, 1991).

Results of BLAST analysis suggest that clones J48 and F41 may beoverlapping and encode novel proteins as both share only limitedsequence identity with two EST clones D79577 and AA060454. Neither cloneexhibits strong identity with any known protein motifs. Similarly,clones i33b and F44a appear to be overlapping cDNA clones. One segmentof both clones is virtually identical to regions within EST cDNA clonesN70566 and F13805. EST clones N70566 and F13805 also overlap. Clonesi33b and F44a do not appear to contain sequence motifs found in anyknown proteins. The products of i33b and F44a are expected to be novel.On the other hand, clones D21 and #23–15 appear to be overlapping andencode a novel protein with some homology to a frog neuronalintermediate filament protein.

Example 2 Characterization of P24

I Sequence analysis

The 936 bp cDNA contained a single 651 bp long open reading frame(ORF)encoding a 217 residue protein P24 with a single transmembrane domain.The P24 protein sequence also includes sites for N-linked glycosylation,protein kinase C phosphorylation, and vesicular transport and sorting(Kyte, J., et al., J. Mol. Biol. 157:105–32 (1982)).

BLAST searches revealed a high degree of sequence similarity withneuronal dendritic proteins, P19 (Wang, H. Y., et al., Mol. Pharmacol.48:988–94 (1995)) and P21 (Yu, P. Y., et al., J. Clin. Invest. 95:304–8(1995); Lefkowitz, R. J., J. Biol. Chem. 273:18677–80 (1998)). P24appears to be a more distant relative of this family as the P24 aminoacid sequence exhibits approximately 37% sequence identity to theseproteins, whereas the sequences of P19 and P21 are approximately 55%identical.

II. P24 Interacts with D1 Receptors

A. Pull-down Assays

For pull-down assays, a 550 bp Nco I-Bgl II fragment from pACT-Calcyonwas subcloned into pET30a (Novagen, Madison, Wis.) to produce theS-Calcyon fusion protein containing residues 93–217 of the predictedCalcyon protein as well as an N-terminal tag composed of the 15 residueS peptide of ribonuclease S (Richards, F. M., et al., The Enzymes. ed.:Boyer, P. D., Academic Press, N.Y., pp. 647–806 (1971)). TheS-β-galactosidase fusion protein was composed of bacterialβ-galactosidase protein tagged at its N-terminus with the S peptide.Fusion proteins were induced in BL21(DE3) E. coli with IPTG, and coupledto S-agarose resin (Novagen, Madison, Wis.) by incubating solublefractions of bacterial lysates with resin for 1 h at RT. Followingincubation, unbound proteins were eliminated by washing the resin threetimes in 50 volumes of 25 mM HEPES (pH 7.4), 50 mM NaCl. Interactionassays were performed with GSTD1 purified as described (Bergson, C., etal., J. Neurosci. 15:7821–36 (1995)) or solubilized lysates of D1 HEK293cells in 25 mM HEPES (pH 7.4), 50 mM NaCl, 10% glycerol, 1% bovine serumalbumin containing 0.5% NP-40. Peptide1 421–435 (SVILDYDTDVSLEKI) (SEQID NO. 3) and Peptide2 (NEDQKIGIEIIKRALKI) (SEQ ID NO. 4) weresynthesized using an Applied Biosystems 430A Peptide Synthesizer usingthe FastMoc procedures and reagents supplied by Perkin-Elmer, AppliedBiosystems division. Peptides were resuspended in 100 mM HEPES, pH 7.4at 1 mg/ml prior to addition to ‘pull-down’ reactions. S-protein boundresin and protein targets were mutated for 2 h at RT, washed twice in 10volumes 25 mM HEPES (pH 7.4), 50 mM NaCl, then resuspended in gelloading buffer (Laemmli, U. K., Nature 227:680–5 (1970)).

B. In vivo Labeling and Immunoprecipitation

Approximately 40 h after transfection, pEGFP or pEGFP-Calcyontransfected D1 HEK293 cells were washed and placed in HBS(phosphate-free media) for 1.5 h prior to addition of [³²p]orthophosphate (135 μCi/ml). 2 h later, cells were treated with 100 nMPMA (Phorbol 12-Myristate 13-Acetate) in HBS for 0.5 h at 37°. Cellswere then washed once in cold PBS and solubilized in mild lysis solution(Cytosignal, Irvine, Calif.) containing protease inhibitor cocktail(Boehringer Manheim) and phosphatase inhibitors, 50 μM NaF and 5 μMEGTA. Lysates were centrifuged for 5 min at 14,000 rpm and thesupernatant was incubated at RT. for 1 h with mouse anti-GFP mab(Clontech, Palo Alto, Calif.) (diluted 1:100), followed by 0.02 volumeof protein A/G agarose slurry for 30 min. The resin was washed twicewith mild lysis solution, and adsorbed proteins eluted in SDS PAGEloading buffer (Laemmli, U. K., Nature 227:680–5 (1970)). Samples wereanalyzed by SDS-PAGE, using a 15% gel (Bio-Rad). The gel was dried andvisualized using a Phosphorimager SF (Molecular Dynamics). Values pixelintensity were obtained by “boxing” the bands and subtracting the lanebackground using Image Quant v. 3.3 software (Molecular Dynamics).

C. Functional Characterization

A series of experiments were then undertaken to functionallycharacterize the interaction between P24 and D1 receptor proteins.D1-HEK293 cells, a HEK293 cell line that expresses hD1 receptors, andpEGFP-P24 expression plasmid were used for these studies. pEGFP-P24encodes P24 protein tagged at its N-terminus by enhanced greenfluorescent protein (EGFP). The N-terminal EGFP tag allowed testing todetermine whether P24 directly associates with D1 DA receptors whencoexpressed in mammalian cells in a “pull-down” assay. D1 receptorsco-immunoprecipitated with EGFP-P24 fusion protein transiently expressedin transfected D1-HEK293 cells, and were “pulled down” by GFP mab. Incontrast, D1 receptors did not co-immunoprecipitate with EGFP, althoughthe GFP mab immunoprecipitated EGFP from pEGFP-C3 transfected D1-HEK293cells.

A family of single transmembrane proteins called receptor activitymodifying proteins (RAMP) has been found to increase the sensitivity ofcalcitonin-like peptide GPCR family to the various endogenous peptideligands. These results imply localization of the P24 to D1-HEK293 cellplasma membranes specifically requires interaction with D1 receptorssince the HEK293 cell line used here endogenously express several othertypes of GPCRs including β₂-adrenergic, muscarinic, purinergic andprostaglandin receptors (Wang et al., J. Biol. Chem., 272:26040–26048(1995)). Regional comparison of relative rates of DA release andre-uptake favor a synaptic mode of DA transmission in caudate, whereasnonsynaptic DA transmission is thought to be the norm in PFC. Antibodiesto a 20 residue segment of P24 were developed to characterize itsinteraction with D1 receptors. Affinity-purified P24 antibodies bound toa strong band of approximately 34K present in microsomal proteinfractions purified from rhesus monkey PFC and caudate putamen, but notspleen. Incubation of antibodies with immunizing peptide conjugated toBSA, but not with BSA alone, prior to immunoblotting, preventeddetection of the approximately 34K band. P24 antibodies reacted with anapproximately 24K PFC microsomal protein digested with PNGaseF,suggesting the 34K band corresponded to P24 protein modified by N-linkedoligosaccarhides. The size of the deglycosylated protein agrees with thepredicted molecular weight of the P24 core protein. The P24 antibodyreactive protein proved resistant to solubilization following treatmentof microsomal fractions with chaotropic agents, including 100 mM NaCO₂,suggesting P24 spans a phospholipid bilayer. Taken together our datasupport the prediction that P24 is an N-linked glycosylated,transmembrane protein.

To pinpoint the binding site for Calcyon, shorter fragments of the 81residue D1 receptor bait, pGBTD1₃₆₅₋₄₄₆, were tested for interactionwith pACTCalcyon by the two-hybrid assay. Deletion of the C-terminal 11amino acids had no apparent effect on the ability of the D1 receptorbait to interact with Calcyon as pGBTD1₃₆₅₋₄₃₅ and pACTCalcyonstimulated lacZ expression when cotransformed into yeast. However,deletion of 26 residues from the C-terminus of the D1 receptor preventeddetectable interaction between pGBTD1₃₆₅₋₄₂₀ and pACTCalcyon. Incontrast, the Calcyon:GAL4 AD fusion protein interacted with D1receptor:GAL4 binding domain fusion proteins, encoded by pGBTD₄₂₁₋₄₄₆and pGBTD1₄₂₁₋₄₃₅, in which the N-terminal 55 residues of the D1 ‘bait’were deleted. These results suggest residues 421–435 of the D1 receptorcomprise a minimal domain sufficient for interaction with Calcyon.

The interaction between Calcyon and D1 receptor was confirmed in‘pull-down’ assays. The predicted Calcyon cytoplasmic domain was fusedto the 15 residue S protein recognition sequence (Richards, F. M., etal., The Enzymes. ed.: Boyer, P. D., Academic Press, NY, pp. 647–806(1971)), and the resulting fusion protein, designated S-Calcyon, wasimmobilized with S-agarose resin. An unrelated fusion proteinS-β-galactosidase, served as a negative control. The ability of theS-tagged proteins to associate with a glutathione-S-transferase-D1(GSTD1) fusion protein containing the 81 residue D1 receptor ‘bait’sequence was tested by incubating GSTD1 with immobilized S-Calcyon andS-β-galactosidase. Immunoblots of proteins eluted from the S-agaroseresins were probed with D1 receptor antibodies.

D1 antibodies bound to a band of approximately 36 K, the size predictedfor the fusion protein, in the positive control lane containing purifiedGSTD1. A D1 antibody reactive band of similar size was present in lanescontaining eluate from immobilized S-Calcyon suggesting the protein‘pulled-down’ by S-Calcyon corresponded to the GSTD1 fusion protein. Incontrast, the immunoreactive band was not present in lanes containingeluate from S-β-galactosidase-bound resin. As coomassie stainingrevealed equivalent levels of the S-tagged fusion proteins bound to theS-agarose resin, the ability of S-Calycon to ‘pull-down’ pGBTD1presumably reflects the higher affinity of D1 receptor C-terminalsequences for associating with S-Calcyon than S-β-galactosidase. Toconfirm the necessity of D1 receptor sequences, pull-down assays wereperformed in the presence of a peptide containing D1 receptor residues421–435 (pep421–435), or an unrelated 17 residue peptide, pep2.Pep421–435, but not pep2, prevented detection of the immunoreactiveGSTD1 band suggesting pep421–435 can block interaction between GSTD1 andS-Calcyon. These results further support a role for D1 receptor residues421–435 in mediating interactions between the D1 receptor and Calcyon.In,addition, the results of these in vitro experiments indicate theCalcyon interacts with D1 receptors through its C-terminal segment.

A human embryonic kidney (HEK) 293 cell line, designated D1 HEK293, thatstably expresses hD1 receptors was used to test the ability of S-Calcyonto interact with full-length D1 DA receptors. Detergent solubilizedlysates of D1 HEK293 cells were incubated with resin-immobilizedS-Calcyon and S-β-galactosidase. Proteins retained by the S-taggedtarget polypeptides were analyzed by immunoblotting with D1 receptorantibodies as described above. Full-length D1 receptor polypeptidemigrates with a molecular mass of approximately 48–50 K in D1 HEK293cell lysates. Bands of similar size were also present in lanescontaining D1 HEK293 cell proteins ‘pulled-down’ by S-Calcyon. Incontrast, immunoblot lanes containing proteins ‘pulled-down’ byS-β-galactosidase were devoid of D1 antibody reactive bands. Takentogether, the ‘pull-down’ studies provide further support for the directphysical interaction between D1 receptor and Calcyon proteins.

III. Distribution of P24

A. General Distribution

A 470 nucleotide Sal I-Bgl 11 restriction fragment of P24 was extracted(Qiagen) from agarose and random primer-labeled (Life Technologies) with[α-³²P]dCTP (Amersham) and purified by a Chroma Spin column (Clontech).The human RNA master blot (Clontech) was prehybridized in ExpressHybsolution (Clontech) containing 0.1 mg/ml sheared salmon testes DNA, andhybridized overnight at 65° C., with probe (6×10⁶ cpm/ml) inprehybridization solution containing 30 mg C₀t-1 DNA (LifeTechnologies), and 0.2×SSC. The filter was washed four times in 2×SSC,1% SDS at 65° C., and two times in 0.1×SSC, 0.5% SDS at 55° C. prior toexposure to Biomax MS film (Kodak) with an intensifying screen at −85°C.

Hybridization of the ³²P-labelled probes to a human RNA dot blotindicated that gene encoding the clone designated P24 was expressed in anumber of the same brain regions and peripheral tissues as the D1receptor. The strongest hybridization signals corresponded to P24 probebound to polyA+ RNA purified from caudate/putamen, frontal lobe,subthalamic nucleus, substantia nigra, and kidney.

B. Distribution of P24 in Rhesus Monkey Brain

The distribution of P24 protein in rhesus monkey brain was determined.P24 antibodies produced immunostaining of neurons in numerous brainregions including the dopaminoceptive caudate nucleus and PFC. P24antibodies labeled cell bodies and dendrites of pyramidal neurons in alllayers of PFC, similar to previous descriptions of D1 DA receptorantibody labeling of the primate cortex (Huang, Y. Y., et al., Proc.Natl. Acad. Sci. USA 92, 2446–50 (1995); Saberan-Djoneidi, D., et al.,J. Biol. Chem 270 1888–93 (1995)).

Three female New Zealand White rabbits were immunized with Keyholdlimpet hemocyanin (KLH) conjugated to a twenty residue peptide(NH₂-QLSSPDQQNFPDLEGQRLNC-COOH) (SEQ ID NO: 5). P24-specific antibodieswere affinity-purified from crude serum using BSA-conjugated peptidecoupled to affigel-15 (Biorad). Perfusion and preparation of braintissue from adult macaque monkeys (Macaca mulatta) was carried out asdescribed by Mrzljak et al., 1996. 40 μm sections were incubated withP24 antibodies for 48 h (4° C.), and processed by the avidin-biotinmethod using horseradish peroxidase with an ABC Elite kit (Vector Labs).For double-labeling experiments, sections were incubated with a cocktailof rat D1 monoclonal antibody (RBI), and rabbit P24 antibodies, washed,and incubated with Cy3-conjugated or FITC-conjugated secondaryantibodies (Jackson Immunoresearch). Sections were viewed with MolecularDynamic confocal argon/krypton laser and Nikon Diaphot 200 microscope,and data collected was collected using Molecular Dynamics Image Spacesoftware, and analyzed as a Ray Model projection of thirty mm sections.

Proteins were prepared from monkey tissues. PNGaseF (Boehringer Manheim)digestion of membrane protein fractions was carried out according to themanufacturer's instructions with 1.0 unit recombinant N-glycosidase F.Crude microsomal protein fractions of transfected HEK293 cells wereisolated. The pellet was resuspended in homogenizing buffer, and proteinconcentrations determined. For immunoblotting, proteins were solubilizedin 2×SDS Page loading buffer (Laemmli, U. K. Nature 227:680–5 (1970)),and separated by SDS-PAGE and transferred to PVDF (ICN Biomedicals)sheets (Towbin, et al., Proc. Natl. Acad. Sci. USA 76:4350–4 (1979)).Immunoblots were incubated with either rabbit antibodies to P24 (1:100)or D1 receptor mab (1:100) (RBI), and processed. Bound antibodies weredetected by enhanced chemiluminescence (ECL) using an ECL plus kit(Amersham) and Hyperfilm (Amersham).

Membrane and soluble proteins were prepared from monkey tissues andstored at −75° C. as described by Mrzljak, L., et al., Nature 381, 245–8(1966). N-glycosidase F (Boehringer Manheim) digestion of 100 μgmembrane protein fractions was carried out according to themanufacturer's instructions using 1.0 unit of recombinant enzyme.Peripheral membrane proteins were dislodged from prefrontal cortexmembrane protein fractions by resuspending microsomal protein pellets in100 mM Na₂CO₃ pH 11, or 10 mM HEPES, pH 7.4, 5 mM EDTA buffer containing500 mM NaCl, 6 M urea, 200 mM Na₂SO₄, 100 mM NaBr, or 100 mM NaIfollowed by centrifugation, and recovery of soluble and sedimentedfractions. For immunoblotting, proteins in loading buffer (Laemmli, U.K., Nature 227, 680–5 (1970)) were separated by SDS-PAGE and transferredto PVDF (ICN Biomedicals) sheets (Towbin, H., Proc. Natl. Acad. Sci. USA76, 4350–4 (1979)). Molecular mass was determined relative to mobilityof Perfect protein markers (Novagen, Madison, Wis.). Immunoblots wereincubated with either rabbit antibodies to Calcyon (1:100) or D1receptor (1:100), followed by horseradish peroxides (HRP)-conjugatedanti-rabbit antibodies (1:25,000) (Jackson Immunoresearch) and processedas described (Bergson, C., et al., J. Neurosci. 15, 7821–36 (1995)). Forblocking experiments, diluted Calcyon antibodies were preincubated with50 μg BSA or BSA conjugated to the immunizing peptide for 30′. Boundantibodies were detected by enhanced chemiluminescence using an ECL pluskit (Amersham).

-   -   1. Results

Immunogold electron microscopy of PFC further revealed P24 in spines ofpyramidal neurons. Brain sections were double labeled with P24 rabbitantibodies and a D1 receptor rat monoclonal antibody (Yung, K. K. L., etal., Neuroscience 65: 709–730 (1995) (mab) to test the possibility thatP24 protein may be expressed in D1 receptor-containing neurons. BoundP24 and D1 antibodies were detected with Cy3-conjugated anti-rabbit IgGand a fluorescent isothiocyanate (FITC)-conjugated anti-rat IgG; or,with FITC-conjugated anti-rabbit and Cy3-conjugated anti-rat secondaryantibodies. Both combinations of secondary antibodies gave similarresults. Overlay of the FITC and Cy3 fluorescent staining in PFCproduced a yellow cellular labeling pattern indicating expression of theD1 receptors and P24 protein in the same population of pyramidalneurons. P24 antibodies also labeled D1 receptor expressing medium spinyneurons in caudate. However, unlike the cell for cell relationshipobserved in PFC, expression of P24 in caudate was restricted to alimited population of D1 receptor mab labeled medium spiny neurons. P24protein's cortical versus caudate expression pattern may reflect aregion specific requirement for association of P24 with D1 receptors.The coexpression of Calcyon and D1 receptors in pyramidal neurons andmedium spiny neurons suggests that physical association of the twopolypeptides could occur in vivo. In addition, the differentialexpression of Calcyon in D1 receptor containing cell populations incaudate and cerebral cortex raises the possibility that Calcyon maycontribute cell-specificity to D1 receptor functions.

The distribution of D1 receptors and Calcyon protein in rhesus monkeybrain were compared. Calcyon antibodies produced immunostaining ofneurons in numerous brain regions including the dopaminoceptiveprefrontal cortex and caudate nucleus. Calcyon antibodies labeled cellbodies and dendrites of pyramidal neurons in all layers of prefrontalcortex, in a manner similar to previous descriptions of D1 receptorantibody labeling of the primate prefrontal cortex. In pyramidalneurons, Calcyon antibody labeling of cell bodies is predominantlyassociated with the membranes and vesicles of the Golgi apparatus andmuch more sparsely with the endoplasmic reticulum. Such localization issuggestive of rapid posttranslational assembly of Calcyon and transportfrom the cell body. Immunogold electron microscopy of prefrontal cortexfurther revealed Calcyon protein in small and medium-sized dendrites anddendritic spines receiving asymmetric (excitatory) inputs. Similar to D1receptors (Bergson, C., et al., J. Neurosci. 15:7821–35 (1995)), Calcyonprotein was localized at the periphery of postsynaptic densities indendritic spines, a subcellular location appropriate for association ofthe two polypeptides.

Example 3 Mammalian Cell Expression Studies and cAMP Assay

I. Calcium Imaging and cAMP Assays

For Ca⁺⁺ imaging, cells were rinsed with HBS (150 mM NaCl, 10 mMNaHEPES, 10 mM glucose, 2.5 mM KCI, 4 mM CaCl₂ and 2 mM MgCl₂, pH 7.4)and then loaded with 5 μM Fura-2 AM (Molecular Probes) in HBS at RT.After 20 min, cells were washed three times with HBS. Assays wereperformed at RT in 1.5 ml of HBS. Drugs were prepared in HBS andmanually applied. For EGTA experiments, Ca⁺⁺-free HBS was preparedcontaining 0.25 mM EGTA. Samples were viewed with a Zeiss Axiovert 135microscope (63×objective). Images were collected with a CCD cameraconnected to a Silicon Graphics workstation, and analyzed withInovision-Ratiotool 4.3.5. Transfected cells were identified byillumination at 490 nm, and selected as areas for analysis. Cells weresequentially illuminated for less than 100 ms, first at 340 nm, and thenat 380 nm (Grynkiewicz, G., et al., J. Biol. Chem 260, 3440–50 (1985)).Fluorescence emission at 510 nm was monitored for each excitationwavelength via the CCD camera at 10 s intervals. Average pixelintensities within 6–8 selected areas (each area corresponded to singletransfected cell) for both wavelengths at each time point were digitizedand stored on the computer.

For cAMP assay, cells were washed once with HBS, and then exposed toagonists, DA-HCl (Sigma), SKF-81297-HBr (RBI), ATP (Boehringer Manheim)or isoproterenol-HCI (RBI), and bisindolylmaleimide I HCl (Calbiochem)at 37° in HBS. After timed incubation, plates were placed on ice, mediaaspirated and cells washed once in 1.0 ml cold PBS. After aspiration,cells were lysed by addition of 0.75 ml 0.1 N HCl. Followingcentrifugation to pellet proteins, cAMP levels in supernatant weredetermined by Direct cAMP Enzyme Immunoassay Kit (Assay Designs, Inc.)according to the supplier's protocol. Protein pellets were firstresuspended in boiling 10% SDS, then SDS concentration diluted to 0.9%by addition of 10 mM Tris pH 7.4, and protein concentrations determined(Bradford, M. M., Analytical Biochemistry 72, 248–54 (1976)). cAMPassays were performed in triplicate for each ligand concentration.Results (pmol cAMP/mg protein) are reported as average of threeindependent transfection experiments.

HEK293 cells and D1-HEK293 cells were transfected with pEGFP-P24 DNA tostudy the effects of P24:DI receptor interaction on protein localizationby confocal microscopy. A 0.9 kb Eco RI-Xho I cDNA fragment encodingfull-length P24 was isolated from pACTP24, and inserted into pEGFP-C3(Clontech). Microsomal protein (200 mg) isolated from stably transfectedD1 or D5 receptor HEK293 cell lines was pelleted, and briefly sonicatedin 50 mM Tris (pH 7.4), 200 mM NaCl, 0.5% Triton™ X-100 to solubilizereceptors. An equal volume of S-P22100-217 or S-β-galactosidase proteinhomogenate in the same buffer was combined with the solubilizedtransfected HEK293 cell microsomal fractions, and incubated for 2 h at4° C. prior to addition of 0.05 volume of S-agarose slurry (Novagen).The mixture was nutated overnight to permit complex formation. The resinwas washed three times in 50 mM Tris (pH 7.4), 200 mM NaCl, 0.05%Triton™ X-100, and bound proteins were eluted in SDS Page loadingbuffer, and immunoblotted as described above.

II. Results

The fusion protein appeared to be excluded from nuclei of bothtransfected cell types. In transfected HEK293 cells, fluorescentEGFP-P24 fusion protein exhibited a uniformly cytoplasmic distribution.The EGFP-P24 derived cytoplasmic fluorescence appeared more diffuse inD1-HEK293 cells than in HEK293 cells. In addition, in D1-HEK293 cells,the fluorescent signal outlined the cell's perimeter, presumablyreflecting EGFP-P24 protein localized in the plasma membrane. Incontrast, pEGFP-C3 vector DNA produced a ubiquitous fluorescent signalin both cell types. The dual plasma membrane/cytoplasmic localization ofEGFP-P24 in D1-HEK293 cells suggests D1 receptors play a role intrafficking P24 to the plasma membrane.

Example 4 Effects of Calcyon Expression on D1 Receptor Signaling inHEK293 Cells

It was determined whether Calcyon influenced D1 receptor coupling to thecAMP second messenger system in D1 HEK293 cells transfected withexpression plasmids encoding either enhanced green fluorescent protein(EGFP), or Calcyon tagged at its N-terminus by EGFP. D1 HEK293 cells donot endogenously express Calcyon protein. DA as well as D1 specificagonist, SKF81297 produced concentration dependent saturable increasesin cAMP levels in D1 HEK293 cells transfected with either pEGFP orpEGFP-Calcyon. However, DA stimulated comparable levels of cAMP in bothcell types suggesting that Calcyon expression does not alter D1 receptoractivation of cAMP-dependent signaling pathways.

To test the possibility that Calcyon might play a role in D1 receptorstimulation of IP₃ turnover and resulting mobilization of Ca⁺⁺ _(i)stores, as reported from studies in brain (Wang, H. Y., et al., Mol.Pharmacol. 48:988–94 (1995)) and kidney, D1 HEK293 cells were loadedwith the Ca⁺⁺ indicator dye Fura-2 AM (Grynkiewicz, G., et al., J. Biol.Chem 260:3440–50 (1985)). Changes in Ca⁺⁺ _(i) levels were measuredusing ratiofluorometric imaging following bath application of ligand.Application of DA or 10:M SKF81297 produced no detectable response inuntransfected D1 HEK293 cells, or cells expressing either EGFP orEGFP-Calcyon. In vivo, DA is considered primarily to be aneuromodulator. Therefore, to test whether Calcyon plays a role in theD1 receptor's ability to modulate other stimuli, we applied agonists ofendogenous GPCRs prior to stimulating D1 receptors. Stimulation ofendogenous P2Y purinergic receptors (Wang, Q., et al., J. Biol. Chem.272:26040–8 (1997)) with ATP produced a rise in Ca⁺⁺ _(i) in D1 HEK293cells. SKF 81297, when applied following ATP, also triggered animmediate increase in Ca⁺⁺ _(i in) EGFP-Calcyon expression D1 HEK293cells. The response of EGFP-Calcyon expressing cells to the D1 agonistwas comparable in magnitude, but longer in duration than the responseproduced by ATP. Similar responses were observed in cells bathed inCa⁺⁺-free medium containing the cation chelator EGTA, suggesting theobserved rises in Ca⁺⁺ _(i) likely reflect release of Ca⁺⁺ fromintracellular stores rather than influx of extracellular Ca⁺⁺.Application of the D1 receptor antagonist, SCH23390, prior to ATP,blocked the response to SKF81927 further indicating a requirement for D1receptors. In contrast, SKF81297 produced a gradual, but small increasein Ca⁺⁺ in the untransfected or pEGFP-transfected D1 HEK293 cells.Comparison of the response produced by the D1 agonist in EGFP-Calcyonexpressing cells to cells expressing EGFP indicates that one function ofCalcyon is to enhance the ability of D1 receptors to mobilize Ca⁺⁺ _(i).

EGFP-Calcyon transfected D1 HEK293 cells also responded to SKF81297 whenapplied after stimulating endogenous M1 muscarinic receptors (Wang, Q.,et al., J. Biol. Chem, 272:26040–8 (1997)) with 10 μM carbachol. Incontrast, EGFP-Calcyon transfected D1 HEK293 cells failed to respond toSKF81297 following stimulation of endogenous β₂-adrenergic receptors(Wang, Q., et al., J. Biol. Chem, 272:26040–8 (1997)) with 10 μMisoproterenol, although isoproterenol increased cAMP levels. As M1muscarinic and P2Y purinergic receptors activate G_(q), whereasβ₂-adrenergic receptors activate G_(S), these studies collectivelysuggest that stimulation of a G_(q)-coupled GPCR is important in theability of Calcyon protein to potentiate D1 receptor-stimulated Ca⁺⁺_(i) mobilization. In addition, the combination of ATP and SKF81297,stimulated comparable levels of cAMP accumulation in EGFP andEGFP-Calcyon expressing cells. As such, the burst of Ca⁺⁺ _(i)stimulated by SKF81297 in EGFP-Calcyon expressing cells appears to occurindependent of G_(S) activation of adenylyl cyclase.

The Calcyon sequence contains two consensus protein kinase C (PKC)phosphorylation sites within its predicted cytoplasmic domain. Todetermine if Calcyon is indeed a substrate for PKC, pEGFP-Calcyon andpEGFP-transfected D1 HEK293 cells were metabolically labeled with³²P-orthophosphate and treated with the PKC activator, phorbol12-myristate 13-acetate (PMA). GFP mab immunoprecipated a ³²P-labeledprotein from pEGFP-Calcyon transfected cells with size equal to thatdetected by Calcyon antibodies. A ³²P-labeled protein of similar masswas not present in pEGFP transfected D1-HEK293 cells. Phosphorimageranalysis revealed approximately five times more label incorporated intothe protein immunoprecipitated from PMA treated cells than fromuntreated cells suggesting Calcyon is a PKC substrate.

To test the possibility that PKC may regulate Calcyon function, D1HEK293 cells expressing EGFP-Calcyon were treated with the PKCinhibitor, bisindolylmaleimide 1 HCl (BisI). Ca⁺⁺ mobilization inducedby SKF81297 was considerably attenuated in cells treated with the PKCinhibitor. The response to D1 agonist in BisI-treated, EGFP-Calcyonexpressing cells resembled the D1 receptor response stimulated in theabsence of Calcyon. Likewise, BisI reduced D1 receptor-stimulated Ca⁺⁺_(i) release in EGFP-Calcyon expressing cells treated with carbachol.These results suggest that the ability of Calcyon to potentiate D1receptor-stimulated Ca⁺⁺ _(i) mobilization is regulated by PKC.

PKC inhibition also altered the response of the P2Y receptor. Followingapplication of BisI, ATP stimulated a rise in Ca⁺⁺ _(i) in D1 HEK293cells expressing EGFP-Calcyon that lasted longer than in the absence ofBisI. The protracted P2Y receptor response lasted ˜300–400 sec, similarin duration to the ATP response observed in EGFP expressing cells. Incontrast, Ca⁺⁺ _(i) levels were typically elevated for approximately 100sec following ATP stimulation of EGFP-Calcyon expressing cells. Theseresults suggest that, in addition to potentiating D1 receptor-stimulatedCa⁺⁺ release, Calcyon may also suppress the ability of P2Y receptors tomobilize Ca⁺⁺ _(i). This aspect of Calcyon function also appears to becAMP-independent as cAMP levels stimulated in response to ATP andSKF81297 in BisI-treated cells compared to untreated cells were similar.M1 muscarinic receptors appeared especially susceptible to Calcyonsuppression because, in some instances, the response to carbachol wascompletely blocked by EGFP-Calcyon expression.

Example 5 Isolation of hD5 Receptor Monoclonal Antibody ProducingHybridoma Cell Lines

In a complementary strategy, a panel of D1 and D5 subtype-specificmonoclonal antibodies (Mab) are being developed. These should be usefulin isolating interacting proteins through immunoprecipitationexperiments. They should also prove useful in confirming interactionswith proteins identified in the yeast two-hybrid screen.

I. Characterization of hD5 Receptor Monoclonal Antibodies

Three female BalbC mice were immunized with purified MBPD5, a fusionprotein consisting of hD5 receptor cDNA encoding residues 375–477 fusedto the C-terminus of E. coli maltose binding protein (MBP) (Bergson etal., 1995b). Splenocytes of one immunized mouse were fused with P3U1myeloma cell lines, and plated on 96 well-plates. Hybridomas werescreened for antibody reactivity with GSTD5 fusion protein by ELISA, andsubsequently subcloned and expanded. Specificity of the 1G1 D5monoclonal antibody cell line, 1G1, was determined by Western blot ofSf9 cells harboring baculovirus vector containing hD1 cDNA (BioSignal),(D1Sf9), and CV-1 cells stably transfected with 12CA5 epitope-tagged hD5receptor (Bergson et al., 1995b), (D5 CV-1). Membrane protein fractions(5 μg) were loaded in wells. Proteins were resolved by SDS-PAGE in a gelcontaining 12.5% polyacrylamide, and electroblotted to a PVDF filter.PVDF filter was blocked and reacted with 1G1 Mab, followed bybiotinylated goat anti-mouse IgG (Jackson Immunoresearch). Bound 1G1 Mabwas detected with an ECL kit (Amersham). The positions of Kaleidoscope(Biorad) molecular size markers were determined.

Further characterization of one clone, called 1G1, confirmed that thisMab specifically detects hD5 protein on Western blot, and quantitativelyimmunoprecipitates D5 receptors. 1G1 Mab reacts with a broad band ofapproximately 50–80 kDa present in the D5 CV-1 lane. Immunoreactivity issimilar to what is observed with a rabbit polyclonal D5 receptorantibody. Diffuse, heterogeneously sized bands with mobility greaterthan approximately 52 kDa represent glycosylated D5 receptors (Bergsonet al., 1995b). In contrast, 1G1 exhibits no detectable reactivity withD1 receptors indicating the Mab 1G1 is specific for D5 receptors. Takentogether, the specificity of the 1G1 Mab as well as its ability toimmunoprecipitate D5 receptors makes it a suitable reagent to isolateproteins that interact with D5 receptors by immunoprecipitation and/orimmunoaffinity chromatography.

The CV-1 cell line (D5 CV-1) stably transfected with 12CA5epitope-tagged hD5 receptor cDNA inserted into the pTetSplice expressionvector (Bergson et al., 1995b; Schokett et al., Proc. Natl. Acad. Sci.USA 92:6522–6 (1995) was used to test whether 1G1 Mab immunoprecipitatesD5 receptors. D5 receptor expression can be induced in D5 CV-1 cells byremoval of tetracycline from the cell culture medium (induced D5 CV-1).If cells are grown in the presence of tetracycline D5 receptorexpression is repressed (uninduced D5 CV-1). For theimmunoprecipitation, 1G1 (+1G1 i.p.) or 12CA5 (+12CA5 i.p.) Mabs wereadded to membrane fractions of uninduced or induced D5 CV-1 cellssolubilized in 1% Triton™X-100, and incubated on ice for 1 h. Followingaddition of 1/10 volume of protein A-Sepharose™, reactions wereincubated overnight at 4° C. The next day, protein A-Sepharose™ waspelleted and washed three times in 10 mM Tris, 150 mM NaCl, pH 8.0.Crude membrane fractions from uninduced and induced D5 CV-1, andimmunoprecipitated proteins (+i.p.) were solubilized in Laemmli loadingbuffer at room temperature for 1 h., and loaded in wells as indicated.The proteins were not boiled in loading buffer as GPCRs aggregate whenboiled. Proteins were fractionated by SDS-PAGE in a gel containing 12.5%polyacrylamide, and electroblotted to a PVDF filter. The PVDF filter wasblocked and reacted with 1G1 Mab, followed by biotinylated goatanti-mouse IgG (Jackson Immunoresearch). Bound antibodies were detectedwith an ECL kit (Amersham). D5 receptor is not detectable in uninducedcells with the 1G1 Mab, but is in the induced cells. Similarly, 1G1quantitatively brings down D5 receptors in induced cells, but does notimmunoprecipitate detectable D5 receptor from uninduced cells. 1G1 Mabimmunoprecipitates proteins with mobility equivalent to that of D5receptors expressed in induced D5 CV-1 cells. Proteins of the same sizeare also immunoprecipitated from induced D5 CV-1 cells by the 12CA5 Mabvia the 12CA5 epitope inserted at the N-terminus of the D5 cDNA. A bandof 110 kDa is present in all lanes in which immunoprecipitated proteinwas loaded suggesting it may correspond to unreduced IgG heavy chaindimers. (Immunoprecipitated 1G1 and 12CA5 antibodies should also bedetected by the goat anti-mouse secondary antibody used for ECLdetection.) Consistent with this possibility, boiling of samples inLaemmli buffer prior to SDS PAGE reduces the mobility of this band to 55kDa, the size of heavy chain monomers.

Example 6 Mammalian Cell Culture and Immunocytochemistry

Human embryonic kidney 293 (HEK293) cells were maintained in DMEM media(Sigma) containing 2 mM glutamine, and 10% fetal calf serum. HEK293 celllines expressing D1 or D5 dopamine receptors were established by calciumphosphate transfection (Canfield and Levenson, Biochem. 32:13782–6(1993)) of pLXSN (Clontech) plasmid containing full-length human D1(Zhou et al., Nature 347:76–80 (1990)) and D5 (Grandy et al., Proc.Natl. Acad. Sci. USA 88:9175–9 (1991)) receptor cDNAs. Stabletransfected cell lines were selected in the above media containing 700mg/ml G418 (Life Technologies), and maintained in 250 Ig/ml G418. D1 andD5 receptor expression was confirmed by Western blotting. pEGFP-C3 andpEGFP-P24 plasmid DNAs were transiently transfected into HEK293 celllines using Effectene (Qiagen, Santa Clarita, Calif.) according tomanufacturer's instructions. D1 receptor expression was confirmed byimmunoblotting with receptor antibodies (Bergson, C., et al., J.Neurosci. 15:7821–36 (1995)). For ‘pull-down’ studies, approximately 10⁷D1 HEK293 cells were scraped from dishes, pelleted, and resuspended in25 mM HEPES (pH 7.4), 50 mM NaCl, 10% glycerol, 1% bovine serum albumin(BSA) containing 0.5% NP-40 by brief sonication. Solubilized celllysates were obtained after a 1 h incubation on ice by pelletinginsoluble fractions at 14,000 rpm for 20 min at 4° C.

pEGFP-Calcyon was created by inserting a 0.9 kb Eco RI-Xho I fragmentencoding full-length Calcyon into pEGFP-C3 (Clontech). pEGFP-C3 andpEGFP-Calcyon plasmid DNAs were transiently transfected into HEK293 andD1 HEK293 cell lines using Effectene™ (Qiagen) according tomanufacturer's recommendations. 2.5×10⁵ cells were seeded in 6-wellplates, and transfected with 0.4 :g plasmid DNA/well for Ca⁺⁺ imaging,cAMP, and immunoprecipitation assays.

Example 7 Calcyon Sequesters PIP₂, which is Indicative that CalcyonCould Inhibit M1 or P2Y-Stimulated Ca⁺⁺ _(i) Release

Functional imaging studies in D1 HEK293 revealed that Calcyon enabledthe D1 receptor to stimulate Ca⁺⁺ _(i) release following a priming stepinvolving stimulation of either the G_(q)-coupled M1 or P2Y receptor. Inaddition, it was observed that Calcyon expression decreases P2Y and M1receptor-stimulated Ca⁺⁺ _(i) release in D1HEK293 cells. For example,the P2Y receptor response lasted ˜300–400 sec in EGFP expressing cells(FIG. 3 a), whereas Ca⁺⁺ _(i) levels were typically elevated for lessthan approximately 100 sec following ATP stimulation of EGFP-Calcyonexpressing cells (FIG. 3 b) indicating inhibition of P2Yreceptor-stimulated Ca⁺⁺ _(i) release by Calcyon. The M1 muscarinicreceptor appears especially susceptible to Calcyon suppression because,in some instances, carbachol-stimulated Ca⁺⁺ _(i) release wasundetectable in EGFP-Calcyon expressing D1HEK293 cells (compare FIG. 3c). These results suggested that, in addition to potentiating D1receptor-stimulated Ca⁺⁺ _(i) release, Calcyon may also suppress theability of P2Y or M1 receptors to mobilize Ca⁺⁺ _(i).

BLAST search of the SwissProt database using the predicted intracellulardomain of Calcyon revealed significant similarity between thealanine-rich region of Calcyon and the myristolated alanine-rich Ckinase (PKC) substrate (MARCKS) protein. MARCKS has been shown tosequester the acidic phopholipid phosphatidylinositol-4,5-biphosphate(PIP₂) in a process regulated by PKC phosphorylation (Glaser, et al., J.Biol. Chem 271, 26187–93 (1996)). It seems reasonable to propose thatPIP₂ may also play a key role in the priming of Calcyon-enabled, D1receptor stimulated Ca⁺⁺ release as M1 and P2Y receptor stimulationresults in activation of phospholipase Cβ, an enzyme which converts PIP₂to diacylglycerol (DAG) and IP₃. If, like MARCKS, Calcyon sequestersPIP₂, one might predict that EGFP-Calcyon expression would inhibit M1 orP2Y-stimulated Ca⁺⁺ _(i) release. Data shown in FIGS. 3 a–3 c comparingP2Y and M1 stimulated Ca⁺⁺ _(i) release in EGFP versus EGFP-Calcyonexpressing D1HEK293 cells are consistent with this notion. Reasoningfurther by analogy to MARCKS, if G_(q)-coupled receptor stimulationleads to additional phosphorylation of Calcyon, the liberated pools ofPIP₂ may contribute to the large increase in Ca⁺⁺ mobilized by D1receptor stimulation. Other studies showed that the PKC inhibitorbisindolylmalemide inhibits Calcyon-enabled D1 stimulated Ca_(i) ⁺⁺release.

Tests were conducted to determine whether Calcyon can bind PIP₂ usingthe S-Calcyon fusion protein. S-Calcyon contains the predictedcytoplasmic domain of Calcyon (residues 93–217) preceded by an ‘S’ tag(for coupling to S-agarose resin). S-Calcyon bound to 25 μl of S-agaroseresin was incubated for one hour at room temperature with phospholipidvesicles containing phosphotidylcholine (PC), phosphotidylethanolamine(PE), or PC, PE, and PIP₂. Both types of vesicles were radioactivelylabeled with equivalent amounts of ³H-phosphatidylcholine. Followingincubation, resin was washed three times with ten volumes of buffer (20mM Hepes, pH 7.4), and amounts of bound phospholipid were determined byliquid scintillation counting. Parallel assays were conducted with 25 μlof S-agarose to control for non-specific binding of phospholipid toresin only. Results are reported as percent of input radiolabelprecipitated, bars represent the standard error of the mean of eachvalue determined in triplicate. S-Calcyon bound to resin precipitatedPIP₂ containing vesicles more than five times more effectively thatvesicles composed of PC and PE only (FIG. 4). The presence of PIP₂ inlipid vesicle composition makes a significant difference (p<0.001) inthe ability of S-Calcyon to bind phospholipid. In addition, attachmentof S-Calcyon appears to significantly improve S-agarose resin binding tothe PIP₂ containing vesicles (p<0.001). These results are indicativethat Calcyon does sequester PIP₂, and therefore that EGFP-Calcyonexpression would inhibit M1 or P2Y-stimulated Ca⁺⁺ _(i) release.

Modifications and variations of the methods and materials describedherein will be encompassed by the following claims. References citedherein are specifically incorporated.

1. An isolated polynucleotide comprising: (a) a nucleic acid sequenceencoding the calcyon protein of SEQ ID NO: 2; or (b) a fragment of thenucleic acid sequence of SEQ ID NO: 1 that encodes a fragment of theprotein of SEQ ID NO: 2, wherein said fragment of the protein of SEQ IDNO: 2 is at least 125 amino acids in length.
 2. The polynucleotide ofclaim 1 having the sequence of SEQ ID NO:
 1. 3. The isolatedpolynucleotide of claim 1 having a detectable label attached thereto. 4.The polynucleotide of claim 1 wherein the fragment of SEQ ID NO: 2,comprises amino acids 93 to 217 of SEQ ID NO:
 2. 5. The isolatedpolynucleotide of claim 1, comprising nucleic acids 117 to 767 of SEQ IDNO:
 1. 6. An isolated nolynucleotide consisting of at least 14contiguous nucleotides of nucleic acids 117 to 767 of SEQ ID NO: 1 orthe reverse complement of nucleic acids 117 to 767 of SEQ ID NO:
 1. 7.An isolated polynucleotide encoding a fragment of the protein of SEQ IDNO: 2, wherein said fragment comprises at least 20 consecutive aminoacids of SEQ ID NO:
 2. 8. The isolated polynucleotide of claim 7,comprising nucleic acids 279 to 338 of SEQ ID NO: 1 encoding apolypeptide having the sequence of SEQ ID NO:
 5. 9. A plurality ofisolated polynucleotides, each polynucleotide consisting of at least 14contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 1 orthe reverse complement of SEQ ID NO: 1, wherein each of the isolatedpolynucleotides or the reverse complement of each of the isolatedpolynucleotides do not include any overlapping sequences.